Influence of type of linkage and spacer on the interaction of beta-galactoside-binding proteins with immobilized affinity ligands.
Affinity chromatography provides a powerful tool for isolation of carbohydrate-binding proteins. However, the choice of the ligand and spacer has an important impact on effectiveness. The influence of several different ligands on qualitative and quantitative aspects of the purification of two beta-galactoside-specific lectins has been evaluated. Sepharose was modified by coupling four types of neoglycoproteins (galactosylated or lactosylated bovine serum albumin with increasing sugar content) and two naturally occurring asialoglycoproteins at similar densities. Carbohydrate ligands at essentially equal density were made accessible to the lectins by seven commonly used methods. The yield of mistletoe lectin was high when lactosylated neoglycoproteins were used for separation. For these resins the sugar incorporation exceeded 10 sugar groups per protein carrier molecule. The yield was similarly high with the asialoglycoproteins and with lactose; the sugar was coupled to the resin as a p-aminophenyl derivative or by means of divinyl sulfone activation. An epoxy group in linkages of galactose or lactose decreased the binding capacity. A quantitatively similar degree of protein yields was obtained for the beta-galactoside-binding protein of bovine heart, although different proteins were obtained when neoglycoproteins were used as ligand. The nature of the affinity ligand in lectin purification can increase the yield and may also influence the profile of the carbohydrate-binding proteins.
Gabius
H J
HJ
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
D000818:Animals; D050260:Carbohydrate Metabolism; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D037161:Galectins; D006023:Glycoproteins; D006388:Hemagglutinins / Q000302:isolation & purification* / Q000378:metabolism; D037102:Lectins / Q000302:isolation & purification*; D008024:Ligands; D008970:Molecular Weight; D009206:Myocardium / Q000737:chemistry; D028321:Plant Preparations; D010940:Plant Proteins; D012116:Resins, Plant; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological / Q000302:isolation & purification
D037161:Galectins; D006023:Glycoproteins; D006388:Hemagglutinins; D037102:Lectins; D008024:Ligands; D028321:Plant Preparations; D010940:Plant Proteins; D012116:Resins, Plant; D054790:Ribosome Inactivating Proteins, Type 2; D014118:Toxins, Biological; C009674:ribosome inactivating protein, Viscum